The large-scale, economic purification of proteins is an increasingly important problem for the biopharmaceutical industry. Therapeutic proteins are typically produced using prokaryotic or eukaryotic cell lines that are engineered to express the protein of interest from a recombinant plasmid containing the gene encoding the protein. Separation of the desired protein from the mixture of components fed to the cells and cellular by-products to an adequate purity, e.g., sufficient for use as a human therapeutic, poses a formidable challenge to biologics manufacturers for several reasons.
Manufacturers of protein-based pharmaceutical products must comply with strict regulatory standards, including extremely stringent purity requirements. To ensure safety, regulatory agencies, such as Food and Drug Administration (FDA), require that protein-based pharmaceutical products are substantially free from impurities, including both product related contaminants such as aggregates, fragments and variants of the recombinant protein and process related contaminants such as host cell proteins, media components, viruses, DNA and endotoxins. While various protein purification schemes are available and widely used in the biopharmaceutical industry, they typically include an affinity-purification step, such as Protein A purification in the case of antibodies, in order to reach a pharmaceutically acceptable degree of purity.
Despite the advent of advanced chromatography and filtration methods, affinity chromatography is still often employed as a capture step to meet the purity, yield, and throughput requirements for biopharmaceutical antibody purification in order to achieve therapeutic grade purity. Despite a high binding affinity of Protein A chromatography for antibodies (about 10−8 M for human IgG), and ability to remove as much as 99.5% of impurities, affinity chromatography is an expensive purification step for use in purifying therapeutic proteins on a commercial scale. Not only is Protein A significantly more expensive than non-affinity media, it also has problems such as resin instability, difficulty with cleaning, ligand leakage, and potential immunogenicity of Protein A or Protein A related compounds contaminating the purified product. The high cost and instability of affinity media, however, increases the ultimate cost of protein-based therapeutics, particularly those requiring high doses and/or chronic administration. Chromatography alone can account for two thirds of downstream processing costs and, with respect to monoclonal antibodies, the resin cost for an affinity-capture column can overwhelm raw materials cost. (see Rathore et al, Costing Issues in the Production of Biopharmaceuticals, BioPharm International, Feb. 1, 2004).
Even if Protein A affinity chromatography is used, adequate purity is often not achieved unless several purification steps are combined, thereby further increasing cost and reducing product yield. Since antibodies account for an increasingly large percentage of therapeutic biologics on the market and in development for the treatment of cancer, autoimmune disease, infectious disease, cardiovascular disease, and transplant rejection, there is a need for a process that can purify proteins using fewer steps and thus realizing lower cost.
US Pat. Pub. No. 2003/0229212, which is hereby incorporated by reference in its entirety, describes a method for purifying antibodies from a mixture containing host cell proteins using non-affinity chromatography purification steps followed by a high-performance tangential-flow filtration (HPTFF) step. As determined by the reduction of CHOPs (Chinese Hamster Ovary cell Proteins), that purification process resulted in contaminant levels of about 144,780 ppm CHOPs after cation exchange purification, about 410 ppm CHOPs after anion exchange purification, and about 17-21 ppm CHOPs after HPTFF purification (final step), thereby providing a three-step non-affinity process. The purification step of HPTFF, which uses a charged membrane to separate impurities (without limit to relative size), such as proteins, DNA and endotoxins, and to eliminate protein oligomers and degradation products from the mixture containing the antibodies, was essential to achieve the final purity.
Moreover, HPTFF has disadvantages, namely (1) it is an extra step in the development, optimization and scale-up of the protein therapeutic, which requires membrane cleaning, validation, commercial availability of large-scale GMP cassettes (which is not yet available for HPTFF), and additional buffers, equipment and greater process time, and (2) it increases costs while risking potential loss of product by compromising antibody integrity (by degradation or aggregation or other molecular change that affects molecular activity).
Therefore, it would be desirable to obtain high purity of a protein therapeutic from a two-step, non-affinity process, which is not based on HPTFF, at a reduced cost in comparison with affinity-based purification and other multi-step purification processes. It would be advantageous if the non-affinity purification process could remove host cell proteins, nucleic acids, endotoxins, product-related contaminants, e.g., aggregated, oxidized, deamidated or degraded forms of the protein, and media additives, e.g., lipids, vitamins, insulin, methotrexate, amino acids, carbon sources such as glucose, etc.
The development of a purification scheme applicable to various types of proteins, scaleable, controllable, and that employs cheaper, reusable resins will allow its integration into product development at a very early stage in overall drug development. This approach to the design of a purification scheme can minimize costly changes to manufacturing processes which may otherwise be necessary later in drug development or, worse, after approval. As the process is scaled-up and approaches GMP production conditions, additional inherent complexities arise, including those associated with resin packing and buffer preparation. The manufacturing process, and its capacity, can be improved by simplifying the purification scheme by eliminating process steps and maximizing throughput and productivity, while maintaining the integrity and purity of the molecule that is being purified. Therefore, it would be desirable and advantageous to start with a simple and efficient process that can produce a drug substance of high quality and safety.